In animal cells, the genome is replicated in a regionally programmed manner during S phase, and this timing schedule is correlated with gene expression. Housekeeping genes are uniformly early replicating, while many tissue specific genes replicate late in most cell types, but are developmentally regulated to replicate early in the tissue of expression. Little is known about the biological significance of this epigenetic process or how it is controlled at the molecular level. We will use a transgenic mouse model to decipher how replication origins are set up to initiate DNA synthesis at a specific point in S phase through the formation of local histone modification patterns. These studies should also reveal the mechanism by which these epigenetic structures are maintained through cell division. We have developed a sophisticated microinjection system that measures how DNA is repackaged following replication at any point in S phase and this can be used to directly evaluate how replication timing affects gene expression. Microarray studies should help explain how this global repression mechanism works together with other modalities to bring about a multi-layered closed chromatin structure. Many regions of the genome, including the olfactory and immune receptor genes, are expressed monoallelically and programmed to replicate asynchronously with one allele undergoing DNA synthesis early in S and the other late. We have designed experiments to identify the molecular components, including RNA and proteins which work together to set up this pattern during early development. In addition, targeted and transgenic mice will be used to show that replication timing is a key factor in the control of monoallelic expression and to decipher its molecular mechanism. These studies should clarify how this regulatory system insures that the stochastic choice of genes from a multi gene array is restricted to a single product per cell, thus serving as an important epigenetic mechanism for generating diversity.